In mammalian liver, SCD1 is the main enzyme responsible for the conversion of the saturated fatty acyl-CoAs, stearoyl-CoA (18:0) and palmitoyl-CoA (16:0), to their respective monounsaturated fatty acyl-CoAs, oleyl-CoA (18:1) and palmitoleyl-CoA (16:1) (Figure A). Here we asked whether a diet enriched in saturated fatty acids can still induce hepatic insulin resistance when the hepatic expression and activity of SCD1 is markedly curtailed.
Systemic administration of Scd1 ASO downregulates liver Scd1 expression and activity in OF rats.
Treatment with Scd1 ASO decreases liver Scd1 expression and activity.
Following 2 injections of Scd1 ASO over 5 days (Figure B), liver Scd1 mRNA was decreased by 80%, as determined by quantitative real-time PCR (Q-PCR). Similarly, total SCD activity was decreased by approximately 50% in the liver of rats treated with Scd1 ASO compared with animals treated with scrambled ASO (SCR) (Figure B). The decrease in Scd1 activity was sufficient to alter the total pools of C18 acyl-CoAs, leading to a decrease in the C18 desaturation index, the ratio of C18:1 to C18:0 (oleyl-CoA/stearoyl-CoA) (Figure B). ASOs delivered i.p. were mainly taken up by the liver, but they also reached the peritoneal fat. Indeed, Scd1 mRNA levels were also decreased in epididymal fat pads. Food intake and body weight were not significantly different between the 2 treatment groups (Supplemental Table 1; supplemental material available online with this article; doi:10.1172/JCI26991DS1).
Effects of Scd1 deficiency on lipid metabolism.
Consistent with observations in chronic models of SCD1 deficiency (24
), the levels of circulating triglycerides (TGs) were decreased by SCD1 ASO treatment (data not shown). However, the hepatic concentration of long-chain fatty acyl-CoAs (LCFA-CoAs) and of TGs was increased (Figure C). Consistent with these increases in tissue lipid levels, treatment with Scd1 ASO also activated the JNK pathway in the liver as manifested by increased JNK phosphorylation and activity (Figure C).
Since SCD1 deficiency has been previously shown to be associated with decreases in the hepatic expression of lipogenic genes, we next examined the hepatic expression of fatty acid synthase (FAS), acetyl-CoA carboxylase (ACC), and cholesterol hydroxylase 7α (CYP7A1) and the phosphorylation of AMP kinase (AMPK) and ACC at the completion of the insulin clamp procedure (Figure , A and B) and under basal conditions (Figure , C and D). At the completion of the clamp studies, the short-term downregulation of Scd1 expression had only marginal effects on these liver enzymes (Figure B). Under basal (overnight fast) conditions, treatment with Scd1 ASO moderately decreased the expression of FAS, ACC, and CYP7A1 (Figure D). These findings suggest that increased liver lipogenesis is not likely to account for the increase in liver TG and LCFA-CoA levels in rats treated with Scd1 ASO.
Scd1 deficiency moderately decreases the expression of lipogenic genes in the liver.
Scd1 is required for diet-induced insulin resistance in rats.
In order to examine the impact of Scd1 deficiency on diet-induced insulin resistance, we next performed insulin clamp studies in OF rats (Figure A). Under conditions of physiological hyperinsulinemia, the rates of glucose infusion required to maintain euglycemia were markedly lower in OF rats compared with control rats fed a standard chow (SC) diet (7.9 ± 0.7 versus 15.4 ± 0.2 mg/kg/min; P < 0.01) when SCR was administered (Figure B). On the other hand, when Scd1 ASO was given, the rate of glucose infusion required to maintain euglycemia in OF rats was markedly increased up to the levels observed in control SC rats (15.2 ± 0.4 mg/kg/min; Figure B). Scd1 ASO did not significantly alter the levels of glucoregulatory hormones (Supplemental Table 1) and the rate of glucose uptake (Figure B). Conversely, this intervention markedly suppressed liver glucose production (Figure , B and D), and this effect entirely accounted for its effect on glucose metabolism. Glucose production represents the net contribution of gluconeogenesis and glycogenolysis (Figure C). However, a portion of glucose entering the liver via phosphorylation of glucose is also a substrate for dephosphorylation via glucose-6-phosphatase (Glc-6-Pase, encoded in rodents by the G6pc gene), creating a futile (glucose) cycle (Figure C). To delineate the mechanisms by which Scd1 inhibition modulates liver glucose homeostasis, we estimated the in vivo flux through Glc-6-Pase (Glc-6-Pase flux) and the relative contribution of glucose cycling, gluconeogenesis, and glycogenolysis to glucose output (Figure , D and E). Treatment with Scd1 ASO markedly and similarly decreased the rates of glucose cycling and Glc-6-Pase flux (Figure D) in parallel to its effects on glucose production (Figure D). The decrease in glucose production was accounted for by a marked inhibition of gluconeogenesis as well as glycogenolysis (Figure E and Supplemental Table 2).
Scd1 deficiency negates the effects of OF on hepatic glucose fluxes.
Liver-selective Scd1 deficiency normalizes hepatic insulin action in OF rats.
The i.p. delivery of Scd1 ASO led to a marked decrease in Scd1 expression not only in the liver but also in epididymal adipose tissue. In order to target Scd1 ASO to the liver, we delivered a 4-fold lower dose of ASO as a slow intraportal infusion rather than an i.p. bolus (Figure A). This protocol resulted in a significant decrease in liver expression of Scd1 in the absence of significant changes in Scd1 expression in epididymal fat (Figure B). Under conditions of physiological hyperinsulinemia, intraportal infusion of Scd1 ASO markedly improved insulin’s ability to restrain glucose production (Figure B). To more directly examine whether the decreased expression of SCD1 in adipose tissue is required for the effects of SCD1 ASO treatment on liver glucose fluxes, we compared subgroups of OF rats displaying similar decreases in liver Scd1 expression induced by either i.p. or intraportal delivery of ASO (Figure , C–F). The i.p. administration of Scd1 ASO dramatically suppressed Scd1 expression in epididymal fat pads while the intraportal infusion of the same ASO had no detectable impact on adipose tissue Scd1 expression (Figure , C and E). However, the 2 treatment regimens led to similar and dramatic improvements in hepatic insulin action (Figure , D and F). Thus, the approximately 70% decrease in liver Scd1 expression is sufficient to negate the onset of hepatic insulin resistance in this model.
Selective attenuation of liver Scd1 expression normalizes hepatic insulin action in OF rats.
Scd1 is required for diet-induced insulin resistance in mice.
In order to examine whether Scd1 also plays a critical role in the development of diet-induced hepatic insulin resistance in HF-fed mice, we next performed insulin clamp studies in Scd1-deficient conscious mice (Figure , A–C). Treatment with Scd1 ASO markedly decreased hepatic Scd1 mRNA and protein (Figure B). The enzymatic (desaturase) activity of Scd1 was markedly increased by HF feeding in mice and dramatically lowered by treatment with Scd1 ASO (Figure D). Under conditions of physiological hyperinsulinemia, the rates of glucose production were markedly increased in HF-fed mice compared with control mice fed an SC diet when the SCR was administered (Figure D). Importantly, in HF-fed mice treated with Scd1 ASO, the rate of glucose production was markedly suppressed to levels similar to those of control mice (Figure D). Of interest, Scd1 deficiency did not have an impact on the modest decrease in glucose uptake that was induced by HF feeding (Figure D). It should be noted that Scd1 ASO used in these experiments was directed against an entirely different region of Scd1 mRNA. Overall, our findings indicate that Scd1 deficiency exerts a protective effect against the development of hepatic insulin resistance in HF-fed rats and mice.
Scd1 deficiency normalizes hepatic insulin action in HF-fed mice.
Scd1 deficiency enhances Akt phosphorylation and suppresses phosphoenolpyruvate carboxykinase and Glc-6-Pase expression in OF rats.
These metabolic data indicated that Scd1 ASO treatment enhanced insulin action to an extent that restored insulin sensitivity to normal levels in insulin-resistant animals. Enhanced insulin signaling could account for these effects. Thus, we next assessed the effect of short-term Scd1 deficiency on Akt phosphorylation and on the expression of Glc-6-Pase, encoded by G6pc, and phosphoenolpyruvate carboxykinase (PEPCK), encoded by Pck1, in the liver. Decreased expression of Scd1 led to a marked increase in the hepatic phosphorylation of Akt. Western blot analysis (Figure A) showed increased phosphorylation of Akt at both Ser473 (4-fold; Figure B) and Thr308 (3-fold; Figure B) in animals treated with Scd1 ASO as compared with control (scrambled ASO [SCR]) ASO–treated animals. Hepatic Akt phosphorylation in these OF Scd1-deficient animals was even higher than in animals that are not insulin resistant on a regular chow diet. We next investigated whether these changes in Akt activation may have led to transcriptional modulation of the glucoregulatory enzymes PEPCK and Glc-6-Pase. Q-PCR experiments showed that the expression of PEPCK was suppressed by almost 90% in animals deficient in Scd1 as compared with control animals (Figure C). Similar measurements showed an even more dramatic effect on liver Glc-6-Pase, the expression of which was virtually abolished (Figure C) in Scd1 ASO–treated animals. Thus, it is likely that the decreases in in vivo Glc-6-Pase flux and gluconeogenesis are at least partly due to decreased hepatic expression of these 2 critical glucoregulatory enzymes.
Scd1 deficiency enhances hepatic insulin action in OF rats.
Scd1 deficiency enhances hepatic insulin signaling in OF rats.
In order to investigate the effect of Scd1 deficiency on early insulin signaling events, we next injected overnight-fasted rats with an i.p. bolus of insulin and sampled the livers 5 minutes later (Figure A). We assessed the effects of insulin on the phosphorylation of the insulin receptor (Figure , B and C) and insulin receptor substrate 1 (IRS1) (Figure , B and D) by immunoprecipitation followed by Western blots. We also examined the association of the p85α subunit of PI3K with IRS1 (Figure , B and E). The acute administration of insulin failed to significantly stimulate the phosphorylation of IRS1 and its association with PI3K. However, treatment of OF rats with Scd1 ASO partly restored insulin responses. The protein expression of tyrosine phosphatase 1B (PTP1B) (Figure B) was only modestly decreased in response to Scd1 deficiency, and it may account in part for the increased phosphorylation of IRS1. These results suggest that the treatment of OF rats with Scd1 ASO resulted in a moderate stimulation of liver insulin signaling that could account in part for the increased phosphorylation of Akt.
Scd1 deficiency enhances hepatic insulin signaling in OF rats.